1. Field
The following disclosure relates to a Neq hot-start (HS) DNA polymerase in which inteins of a Neq L fragment and a Neq S fragment derived from Nanoarchaeum equitans are linked, and more particularly, to development of mutant Neq HS DNA polymerases having a highly improved PCR amplification rate.
2. Discussion of Related Art
Deoxyribonucleic acid (DNA) polymerases (Enzyme Commission (E.C.) number 2.7.7.7) are enzymes which synthesize a DNA sequence complementary to a template DNA strand in a 5′→3′ direction, and play the most important role in DNA replication or repair in living organisms. The DNA polymerases may be classified into at least six families (families A, B, C, D, X, and Y), based on their amino acid sequences. Most of the DNA polymerases belonging to the family B can initiate replication with high fidelity since they have a 3′→5′ exonuclease activity referred to as proofreading activity. With the development of PCR techniques using thermostable DNA polymerases, attention has been directed to thermostable DNA polymerases. Thus, various thermostable DNA polymerases from thermophiles and hyperthermophiles have been developed. In particular, thermostable DNA polymerases from hyperthermophilic archaea such as Thermococcus litoralis, Pyrococcus furiosus and the like have been used in PCR requiring high-fidelity amplification since such thermostable DNA polymerases have a 3′→5′ exonuclease activity referred to as proofreading activity as well as a DNA polymerization activity.
An intein is a protein insertion sequence that is present within a precursor protein sequence. Since an intein sequence is removed from a precursor protein through a self-splicing process, such intein sequence does not affect the structure and activities of the final protein made from the precursor protein. Protein splicing is a process occurring after translation of proteins. During this process, the intein sequences are consistently removed from the precursor protein by means of a self-splicing action, and extein—domains constituting the final protein exhibiting activities—are linked to each other through a normal peptide bond in the process.
Nanoarchaeum equitans is a nano-sized anaerobe initially isolated from a submarine hot vent at the Kolbeinsey ridge in Iceland. This strain is a living organism that parasitically grows on the surface of a specific host, Ignicoccus sp. strain KIN4/I, under strict anaerobic conditions.
It was reported that Neq DNA polymerase is present in the N. equitans genome and is composed of two genes, separated by 83,295 bp, coding for the Neq DNA polymerase. That is, the DNA polymerase is coded by an extein-coding region and a split mini-intein-coding region. Neq DNA polymerase is produced by two genes which code for a large fragment (Neq L) and a small fragment (Neq S) of the Neq DNA polymerase. That is, polypeptides are expressed from each of the two genes which are separately present on the genome, and are linked by a peptide bond through protein trans-splicing, thereby yielding an active DNA polymerase. The large fragment (Neq L) of the Neq DNA polymerase consists of an extein domain composed of 578 amino acid residues, and an intein domain composed of 98 amino acid residues, which participates in the protein trans-splicing, and corresponds to an amino-terminal part (N-terminal part) of the Neq DNA polymerase (Korean Patent No. 10-0793007 and U.S. Pat. No. 7,749,732). Also, the small fragment (Neq S) of the Neq DNA polymerase consists of an intein domain composed of 30 amino acid residues, and an extein domain composed of 223 amino acid residues, and corresponds to a carboxyl-terminal part (C-terminal part) of the Neq DNA polymerase. The genes coding for the large fragment and the small fragment of the Neq DNA polymerase were cloned into one expression vector, and expressed in Escherichia coli. Then, the E. coli strain was collected, and homogenized by sonication. Thereafter, it was confirmed that a trans-splicing reaction occurred at a high temperature through SDS-PAGE and enzymatic activities. That is, a protein in which inteins were removed through protein trans-splicing at a high temperature and having only exteins linked through a peptide bond was designated Neq C (in the protein trans-spliced form of Neq DNA polymerase). Also, a DNA polymerase produced by recombining an extein-coding region of the Neq L fragment gene, from which an intein-coding region was removed, with an extein-coding region of the Neq S fragment gene, from which an intein-coding region was removed, and expressing the recombinant as a single polypeptide chain was designated Neq P (in a genetically protein splicing-processed form of Neq DNA polymerase). It was reported that the Neq C and Neq P were prepared through different methods, but were enzymes exhibiting the same activities and biochemical characteristics.
Also, when the recombinant vectors expressing the Neq L and S fragments were constructed, and the Neq L and S fragments were expressed in E. coli, purified, and added together, it was found that a trans-splicing reaction occurred at a high temperature through SDS-PAGE and enzymatic activities (Korean Patent No. 10-0793007; and U.S. Pat. No. 7,749,732). Also, it has been reported that each of the Neq L and S fragments was purified, and applied to hot-start PCR, based on the fact that the Neq L and S fragments were trans-spliced at a high temperature (Korean Patent No. 10-1230362).
The N-terminal domain of an archaea-derived family-B DNA polymerase contains a specialized pocket that discriminates the deaminated bases such as uracil and hypoxanthine (Fogg M. J. et al., 2002, Nat. Struct. Biol. 9: 922-927; Gill S. et al., 2007, J. Mol. Biol. 372: 855-863). This specialized pocket scans for the presence of uracil; and, on encountering uracil, DNA synthesis is stalled. However, the Neq DNA polymerase has a different structure than the other family-B DNA polymerases. The Neq DNA polymerase is an archaea-derived family-B DNA polymerase that has no pocket recognizing a uracil base and thus can successfully utilize deaminated bases. In this regard, a method of preparing a Neq-plus DNA polymerase-which is a combination of Neq DNA polymerase and Taq DNA polymerase—and PCR applications using uracil-DNA glycosylase (UDG) and dUTP have been reported recently (see Choi J. J. et al., 2008, Appl. Envirn. Microbio. 74: 6563-6569).
As a method of preventing occurrence of crossover contamination in PCR, Longo M. C. et al. suggested a method of performing PCR using dUTP instead of dTTP (Longo M. C. et al., 1990, Gene 93: 125-128). Also, PCR methods, which include treating template DNA with UDG in order to remove a trace amount of contaminated uracil-containing DNA in a sample before initiation of PCR, and inactivating the UDG through heating, and performing PCR using dUTP instead of dTTP, have been reported (Rys P. N. and D. H. Persing. 1993. J. Clin. Microbiol. 31: 2356-2360). As a result. PCR products which are treated with UDG during a PCR procedure or include UDG tend to be currently commercially available.
In recent years, one of the most important techniques in the PCR-related industries is a hot-start (HS) PCR. HS PCR has been used in various fields such as identification of infectious diseases (e.g. HIV), amplification of DNA with low purity, real-time PCR, one-step RT-PCR, etc., and various studies of enzymes associated with the HS PCR have also been conducted. HS PCR is a PCR method in which DNA polymerase activities are inhibited at a low temperature in a procedure of mixing PCR reaction components or an initial PCR denaturation procedure. But DNA polymerase activities are allowed at a temperature greater than or equal to a primer annealing temperature (approximately 55 to 65° C.). That is, in typical PCR procedures non-specific primer binding takes place when a temperature increases during a procedure of mixing PCR components and an initial PCR denaturation procedure. In this case, undesired PCR products are produced by the activities of the polymerase, and thus the undesired PCR products compete with PCR products of interest in a subsequent PCR reaction and interfere with detection of the PCR product of interest. This non-specific amplification is an especially severe barrier in aspects of detecting target DNA present in a low number of copies, amplifying a low concentration of a DNA sample, and performing multiplex PCR using various primers at the same time. HS PCR was developed to avoid undesired PCR products produced by non-specific priming during this initial PCR procedure. In this case, since the DNA polymerase is active at a temperature greater than or equal to a primer annealing temperature, it is possible to enhance specificity of the PCR products.
An HS PCR method that has been used is a manual method. This method is to add one of the components necessary for PCR (for example, MgCl2, Taq DNA polymerase, dNTP, and the like) at an elevated temperature at the beginning of the PCR procedure. However, the method has various problems in that it cannot be used when there are a large number of samples to be treated. A method subsequently developed includes separately preparing main components of PCR using wax and performing PCR while mixing the separately prepared components and melting the wax through heating. This method has problems in that the wax should be melted and added, and may serve as a barrier in separating the PCR products after a PCR reaction, and a total amount of a reaction solution may be increased by addition of the wax. Another method which was the most commercially successful and has been used by some companies such as Invitrogen is a method using an antibody against Taq DNA polymerase. The method may have an effect of inhibiting the activities of the polymerase since the antibody reacts with the enzyme at room temperature, and PCR proceeds due to the activities of the enzyme since the antibody is denatured due to a gradual increase in temperature, and thus is separated from the enzyme. That is, since an increase in temperature allows primers to bind to target DNA at an accurate position, only the target DNA of interest can be specifically amplified. However, this method has problems in that it requires an excessive amount of the antibody, and the antibody is also very expensive.
Still another method developed is a method using a chemically modified DNA polymerase. This technique was developed separately by Roche (U.S. Pat. No. 5,677,152) and Qiagen (U.S. Pat. No. 6,183,998), and has approximately 68% of the HS PCR market share in the U.S. In this method, the Taq DNA polymerase is inactive due to chemical modification, but becomes active again through an initial reactivation procedure (at 95° C. for 10 minutes) of the PCR reaction, thereby enabling PCR. However, this method also has problems in that only approximately 30% of the enzyme is reactivated at an initial stage of the PCR reaction, and it is impossible to amplify a long DNA sequence due to depurination of the template DNA upon reactivation at a high temperature. In spite of the problems of the method, the chemically modified enzyme is currently being used due to convenience of use. Other methods include a method of specifically designing heat-activated primers (Lebedev A. V. et al., 2008, Nucleic Acids Research 36: No. 20 e131), a magnesium precipitation method (see Barnes W M and Rowlyk K R. Molecular and Cellular Probes 16: 167-171) (using a high concentration of Mg, but it is impossible to use Mg at an accurate concentration), the use of pyrophosphatase and pyrophosphate (Bioneer, Korean Patent Application No. 10-2007-01090055), and the like. None of these methods was very successful since they all have critical problems.
There has been a demand for development of new techniques by which HS PCR can be performed effectively at a low manufacturing cost. Based on the fact that a trans-splicing reaction takes place at a high temperature when Neq L and Neq S fragments of the Neq DNA polymerase (both of which contain inteins) are added together, the present inventors have applied the trans-splicing reaction to HS PCR for the first time so as to satisfy these requirements (Korean Patent No. 10-1230362; and US Patent Publication No. 2012/0135472). That is, the HS PCR method referenced above is based on a new concept for explaining that a DNA polymerase has no activities since protein trans-splicing does not occur at a low temperature. But inteins are removed through trans-splicing at a high temperature (60° C. or more; an optimal temperature of 80° C.) and only exteins are linked by means of a peptide bond to form an active Neq DNA polymerase (Korean Patent No. 10-1230362). However, in such method, the Neq L and Neq S fragments should be separately purified, and should be added to a PCR reaction solution at accurate concentrations. Also, in the method, a PCR amplification rate is slow since wild-type Neq L and Neq S fragments are used.